Since a cardiomyocyte loses a proliferative ability in an adult body, it is necessary to conduct heart transplantation in treating a serious heart disease such as cardiac infarction or cardiomyopathy. However, currently, since insufficient donor hearts are available, there is now a pressing need to develop a method of treatment other than heart transplantation.
On the other hand, the recruitment of the ex vivo produced cardiomyocytes is expected to be a most promising method of providing relief for patients in need of heart transplantation. Various methods of preparing cardiomyocytes have been investigated, such as a method of differentiating stem cells (embryonic stem cells or various adult stem cells) into cardiomyocytes or a method for isolating cardiomyocytes from fetuses.
The differentiation of the cardiomyocytes from the embryonic stem cells are positively induced through formation of a cell mass (an embryoid body) by eliminating from culture medium differentiation suppression factors (such as feeder cells, leukemia inhibitory factor: LIF) in the case of mouse embryonic stem cells or differentiation suppression factors (such as feeder cells, basic fibroblast growth factor: bFGF, transforming growth factor: TGF) in the case of human embryonic stem cells.
A mode of in vitro differentiation partially follows a mode of physiological development. Especially, relating to the events of early development, there are a number of commonalities between the mode of physiological development in fertilized egg cells and the mode of in vitro differentiation. In their vitro cardiomyocyte differentiation course, as is also in physiological development, undifferentiated mesoblast cells are first generated, a part of which is changed to programmed cardiomyocytes (pre-cardiac mesoblast cells) and then differentiated into the cardiomyocytes. However, since the embryonic stem cells can differentiate into any types of cells which construct an organ within a body, it is technically difficult to differentiate the embryonic stem cells into only a single type of cell.
Further, since it is also difficult under non-physiological condition (in vitro) to induce differentiation of the embryonic stem cells into all types of cells, there partially remains the undifferentiated cells. Moreover, the mesenchymal stem cells present in bone marrow or umbilical cord and tissue stem cells present in various kinds of tissues (such as neural stem cell, adipose derived stem cells, and skeletal muscle stem cells) are considered to be the adult stem cells, which are considered to have an ability to differentiate into the cardiomyocytes. These cells are believed to differentiate not only into the cardiomyocytes but also into various kinds of cells. Though the details of differentiation mechanisms from any of the adult stem cells to the cardiomyocytes have not been fully elucidated, it is known that these cells form the cardiomyocytes, other differentiated cells, and a cell population containing undifferentiated cells after undergoing a certain period of transition phase.
In summary, all stem cells cause some common deleterious characteristics for clinical application that there are cells other than the cardiomyocytes which are generated from the stem cells as a by-products or undifferentiated cells. Since the undifferentiated cells have a proliferative activity and have an ability to differentiate into many types of cells, a cell population containing the cardiomyocytes generated by differentiation induction can not be transplanted into a living body in the therapy.
Therefore, to safely implement the treatment using stem cells and achieve an ideal treatment effect, it is necessary to develop a method for purifying the cardiomyocytes from the cell population.
To date, the cardiomyocytes have been purified by a method for purifying the cardiomyocytes by specifically expressing a fluorescent marker such as GFP in the cardiomyocytes and selecting a cell expressing a fluorescent marker using cell sorter (Non-patent document 1) or a method for purifying the cardiomyocytes by specifically expressing an antibiotic resistant protein in the cardiomyocytes and selecting the cells using the antibiotic (Non-patent document 2). However, since these methods have to involve in a genetic alteration, which cause an issue relating to the safety, these methods can not be used to prepare the cardiomyocytes for transplantation in the clinical field. Further, since these methods involve in a genetic alteration, an ethical issue and some unpredictable serious risk such as the change in the rate of transformation are associated with a genomic alteration (Non-patent document 3),
It is known in the art that the heart can use a lactic acid generated by a tissue other than the heart (such as skeletal muscle) as an energy source (Non-patent document 4). However, there are no prior art to attempt to purify cardiomyocytes using this feature.
Also, in the heart, the liver, and the kidney, an aspartic acid and a glutamic acid are used for transporting NADH into mitochondria, the mechanism of which is different from that of the other tissue (Non-patent document 5). The transportation of NADH into mitochondria is indispensable for an energy production in mitochondria. However, there are no prior art to attempt to purify cardiomyocytes using this difference in this mechanism.    Non-patent document 1: Muller M, et al., FASEB J. 2000; 14: 2540-2548    Non-patent document 2: Klug MG, et al., J. Clin. Invest. 1996; 98: 216-224    Non-patent document 3: Schroder AR, et al., Cell. 2002; 110: 521-529    Non-patent document 4: Khairallah M, et al., Am J Physiol Heart Circ Physiol 2004; 286, H1461-1470    Non-patent document 5: Chatham J C, et al., J Biol Chem 1995; 270: 7999-8008